Characterisation of a RhoBTB3 deficient mouse model Julia Babette Lutz Dipl. Biol. (University of Düsseldorf) PhD by Thesis The University of Hull and the University of York Hull York Medical School October, 2012 Abstract RhoBTB proteins are atypical members of the Rho-family of small GTPases with a characteristic domain architecture: a GTPase-domain is followed by a proline-rich region, a tandem of two BTB domains and a C-terminal region. RhoBTB3 is the most divergent member of the RhoBTB family and has been shown to have ATPase instead of GTPase activity. Its precise roles at a cellular and organismal level are unknown. In order to investigate the functions of RhoBTB3 in vivo a RhoBTB3 knockout mouse was characterised in this study. The expression pattern of rhobtb3 was studied with the help of a β-galactosidase reporter gene and confirmed in a number of cases by studying protein expression. The overall strong expression in embryos was highest in bone, cartilage, smooth muscle and heart followed by skeletal muscle, skin and localised areas of the nervous system. The expression declines during late development but remains visible in cartilage, heart, kidneys, smooth muscle cells of blood vessels, testes and ovaries of adult animals. Important phenotypic changes in RhoBTB3 knockout animals are reduced survival as well as growth and fertility defects. Testes of knockout animals are smaller than wildtype controls. Proliferation of primary lung fibroblasts and the integrity of the intestinal tract appear not to be affected by knockout of RhoBTB3. RhoBTB3 appears to have a completely novel role in haemostasis and platelets of RhoBTB3 knockout animals have defects in aggregation stimulated by collagen and thrombin as well as in adhesion to fibrinogen and collagen. They express higher numbers of α2β1 and αIIbβ3 integrins which might relate to the molecular origin of the observed defects. Platelet morphology and in vivo tail bleeding times were appeared unaffected by loss of RhoBTB3. A two-hybrid screening yielded Kindlins as potential binding partners of RhoBTB3 and the interaction has been verified in this study by co-immunoprecipitation. Kindlins are mediators of integrin activation and have been implicated in various human diseases of the skin, the intestine and haematopoietic tissue. Page | 2 Table of Contents Abstract ................................................................................................................... 2 List of Illustrations ................................................................................................... 6 Acknowledgements ............................................................................................... 10 Author’s declaration .............................................................................................. 11 Chapter 1 – Introduction ....................................................................................... 12 1.1 The Ras superfamily of small GTPases ................................................... 12 1.2 Rho GTPases .......................................................................................... 13 1.3 RhoBTB proteins ..................................................................................... 15 1.3.1 The structure of RhoBTB proteins ..................................................... 16 1.3.2 Genomic organisation and expression of RHOBTB genes ................ 18 1.4 Functions of RhoBTB proteins ..................................................................... 19 1.4.1 RhoBTB and the actin filament system ............................................. 19 1.4.2 RhoBTB in cell growth and apoptosis ................................................ 19 1.4.3 RhoBTB and vesicle transport ........................................................... 20 1.4.4 RhoBTB and proteasomal degradation ............................................. 22 1.5 RhoBTB proteins in human diseases ....................................................... 23 1.6 Interaction partners of RhoBTB3 ............................................................. 27 1.6.1 The Kindlin family .............................................................................. 28 1.6.2 Expression profile of Kindlins ............................................................ 30 1.6.3 Kindlin related diseases .................................................................... 30 1.7 Mouse models of Rho GTPase knockout .................................................... 31 1.8 Aims of this study ......................................................................................... 35 Chapter 2 - Materials and Methods ....................................................................... 36 2.1 Materials ...................................................................................................... 36 2.1.1 Animals .................................................................................................. 36 2.1.2 Cell lines, bacteria and yeast strains ..................................................... 36 2.1.3 Vectors .................................................................................................. 37 2.1.4 Plasmids ................................................................................................ 37 2.1.5 Oligonucleotides for PCR ...................................................................... 38 2.1.6 Antibodies and fluorescent dyes ............................................................ 39 2.1.7 Chemicals and consumables ................................................................. 40 Page | 3 2.1.8 Characterisation of a RhoBTB3 antibody .............................................. 41 2.2 Methods ....................................................................................................... 45 2.2.1 Molecular Biology .................................................................................. 45 2.2.2 Protein Biochemistry .............................................................................. 50 2.2.3 Haematology ......................................................................................... 53 2.2.4 Histology and immuno-histochemistry ................................................... 58 2.2.5 Microscopy and image processing ........................................................ 61 2.2.6 Cell culturing, transfection and proliferation assay ................................ 62 2.2.7 Microbiological methods ........................................................................ 65 Chapter 3 – Expression analysis of mouse rhobtb3 .............................................. 68 3.1 Introduction .................................................................................................. 68 3.1 Verification of the rhobtb3 knockout and genotyping of mice ....................... 69 3.2 Gene expression of rhobtb3 in mouse embryos .......................................... 71 3.3 Gene expression of rhobtb3 in the adult mouse ........................................ 101 3.4 Protein expression of RhoBTB3 in embryonic tissue ................................. 114 3.5 Discussion ................................................................................................. 124 Chapter 4 - Phenotyping of a RhoBTB3 deficient mouse model ......................... 130 4.1 Introduction ................................................................................................ 130 4.2 Breeding and general observations ........................................................... 131 4.2.1 Difficulties in establishing a RhoBTB3 knockout colony ...................... 131 4.2.3 RhoBTB3 knockout mice have a growth defect ................................... 132 4.2.4 RhoBTB3 knockout males have small testes ...................................... 133 4.2.5 RhoBTB3 knockout lung fibroblasts proliferate normally ..................... 135 4.3 Histological examination of the intestine .................................................... 136 4.3.1 Introduction .......................................................................................... 136 4.3.2 RhoBTB3 knockout mice have normal morphology of the intestine .... 137 4.3.3 RhoBTB3 knockout animals show normal cytoskeletal organisation in the intestine .................................................................................................. 141 4.3.4 RhoBTB3 knockout mice have normal cell proliferation and apoptosis rate in the intestine ....................................................................................... 144 4.4 rhobtb3 deficiency does not affect the expression of other genes ............. 147 4.5 Discussion ................................................................................................. 149 4.5.1 Reduced viability and growth defects in RhoBTB3 knockout mice ...... 149 4.5.2 RhoBTB3 knockout males have a fertility defect and smaller testes ... 150 Page | 4 4.5.3 RhoBTB3 knockout mice have normal intestinal homeostasis ............ 152 4.5.4 rhobtb3 deficiency does not affect the expression of other genes ....... 153 Chapter 5 – Impaired platelet function in RhoBTB3 knockout mice .................... 156 5.1 Introduction ................................................................................................ 156 5.1 Haematological characterisation of RhoBTB3 deficient mice .................... 158 5.2 Platelets from RhoBTB3 knockout mice have an aggregation defect ........ 160 5.3 RhoBTB3 deficiency leads to impaired platelet adhesion to collagen and